For a variety of clinical reasons it is desirable to quantitatively assay one or more phenylethanolamines found in plasma or in urine. For example, the diagnosis of pheochromocytoma has been based upon measurement of the increased excretion of norepinephrine and its metabolite normetanephrine. Such diagnosis is important because these tumors produce a potentially curable form of hypertension, which otherwise is fatal. Also, norepinephrine is the neurotransmitter released from sympathetic nerve terminals. The determination of normetanephrine metabolite can provide important information regarding sympathetic nerve function and norephinephrine metabolism. Another phenylethanolamine of interest is octopamine, the determination of which may assist in the evaluation of encephalopathy.
Unfortunately, techniques for assaying phenylethanolamines and their metabolites are often non-specific, laborious, tedious and/or frequently give false positive results. Henry et al in "A Sensitive Radioenzymatic Assay for Norepinephrine in Tissues and Plasma", Life Sciences, Vol. 16, p. 375 describe the reaction of a phenylethanolamine with tritiated S-adenosyl-L-methionine promoted by the transfer enzyme phenylethanolamine-N-methyl transferase to form the tritiated N-methyl derivative of the phenylethanolamine. Although the method is applied by Henry et al to normetanephrine in a comparison of substrate specificity, they are not successful in detecting normetanephrine. In a related procedure Molinoff et al in "An Enzymatic Assay for Octopamine and Other .beta.-Hydroxylated Phenylethylamines", The Journal of Pharmacology and Experimental Therapeutics, Vol. 170, 1970, No. 2, P. 253 react octopamine with S-adenosyl-L-methionine made radioactive with C.sup.14, also using phenylethanolamine-N-methyl transferase to initiate the transmethylation. Molinoff et al also apply the procedure to normetanephrine to determine the specificity of the assay and they too had no success in assaying normetanephrine. These failures to quantitatively determine normetanephrine have been the subject of speculation. It has been hypothesized that both urine and serum contain an unidentified inhibitor for the phenylethanolamine-N-methyl transferase. See in this regard Manghani et al in "Urinary and Serum Octopamine in Patients with Portal-Systemic Encephalopathy", The Lancet, Nov. 15, 1975, p. 943. Manghani et al describe a method similar to that of Molinoff et al in assaying octopamine in urine. They ascribe poor recovery in undiluted samples to the presence of a phenylethanolamine-N-methyl transferase inhibitor. Some degree of success in determining urinary normetanephrine was achieved by Vlachakis and DeQuattro in "A Simple and Specific Radioenzymatic Assay for Measurement of Urinary Normetanephrine", Biochemical Medicine, 20, pp. 107-114 (1978). However, even here the results are not as satisfactory as desired, there still being too much inhibition.
The present disclosure described a rapid, specific and inexpensive assaying method for the quantitative measurement of two specific phenylethanolamines: normetanephrine and octopamine. The method has particular importance with respect to the detection of normetanephrine in hypertensive patients even during therapy, as no anti-hypertensive drugs interfere with the assay. Furthermore, the method can be conducted without the need for preservatives and with samples, such as blood, collected under less stringent conditions of temperature than that required for the assaying of other phenylethanolamine such as norepinephrine; these advantages arise from the general high stability of normetanephrine and octopamine.
More specifically, a method is provided which is an improvement upon the prior art method of converting normetanephrine to metanephrine as exemplified by the Vlachakis and DeQuattro method, supra, utilizing techniques from the Henry et al method, supra and also from the method of Peuler and Johnson, "Simultaneous Single Isotope Radioenzymatic Assay of Plasma Norepinephrine, Epinephrine and Dopamine", Life Sciences, Vol. 21, p. 625 (1977). In the Peuler and Johnson method, one of the hydrogen atoms of the amine substituent of the phenylethanolamine is replaced by a methyl group, supplied by transmethylation with S-adenosyl-L-methionine promoted by the transfer enzyme phenylethanolamine-N-methyl transferase. The transferable methyl group on the S-adenosyl-L-methionine is tritiated, thereby radioactively labeling the produced N-methylated phenylethanolamine. The present invention provides an improvement on that method achieved in its most preferred form by a combination of the following steps and conditions: (a) whereas the prior art conducts the transmethylation incubation at a pH of about 6.5 to 8.2, in the present invention transmethylation incubation of the mixture is conducted at a pH of at least 8.6, preferably in the range of 8.8-9.3; (b) when the sample is plasma or brain tissue or other substance containing a high level of protein, prior to incubation, the sample is deproteinized; and (c) prior to incubation, the phenylethanolamine in the sample may be concentrated.
The above conditions and steps significantly decrease enzymatic inhibition and sufficiently increase sensitivity of the assay to determine plasma levels of normetanephrine. Octopamine can be determined without the deproteinization and concentration steps but by combining all three steps and conditions, one obtains a greater degree of reliability in the determination of octopamine.
In particular embodiments, the incubation mixture comprises a combination of ethylene diamine tetraacetic acid (EDTA) and a mixture of tris(hydroxymethyl)amino methane and its hydrochloric salt (the mixture being referred to hereinafter as tris) at a molar ratio sufficient to yield the desired pH. For example, by using 25 millimoles of EDTA per mole of tris, a pH of 10.3 is obtained. Deproteinization can be accomplished by reacting protein in the sample with trichloracetic acid and separating the reaction product from the sample. The normetanephrine or octopamine can be purified by weak cation exchange, e.g. conducted by chromatographic extraction through weak cation exchange resin.
The techniques presented by this disclosure are very convenient both to the patient and to laboratory personnel. For example, urine or plasma can be collected in any clean container without any preservative and an aliquot can be stored in a freezer until analysis. The assay procedure allows collection of a random sample when the patient is hypertensive or symptomatic; analysis can be conducted even several days later. The assay is sensitive enough to be used as a routine test. For example, one can measure as little as 30 picograms per milliliter of normetanephrine, thus providing a specific, rapid and accurate assay for the detection of pheochromocytoma.